The present application relates to amino acid sequences comprising epitopes of adenyl cyclase-haemolysin from Bordetella. Adenyl cyclase-haemolysin (AC-Hly) is one of the toxins participating in the Bordetella infectious syndrome. AC-Hly is a bifunctional protein having an adenyl cyclase activity and a haemolytic activity. It is secreted by the bacterium. Its structural gene has been cloned and sequenced (Glaser P. et al., 1988, Molec. Microb. 2, 19–20). It is the case that this protein is part of the family of toxins termed “RTX” for “repeats in toxins” and exhibits homologies with haemolysin from Escherichia coli and Actinobacillus pleuropneumoniae, and the leucotoxins from Pasteurella haemolytica and Actinobacillus actinomycetemcomitans. This protein, like PTX (pertussis toxin), is capable of penetrating into eucaryotic cells such as the macrophages, of being activated by calmodulin, of synthesizing large quantities of cAMP and of disrupting cellular functions (Coote J. 1992. FEMS Microbiol. Rev. 88:137–162).
The inventors have identified, within this sequence, various domains having the capacity to induce the formation of protective antibodies against an infection by Bordetella, in particular by B. pertussis and/or B. parapertussis and/or B. bronchiseptica. 
The subject of the invention is therefore amino acid sequences capable of entering into the composition of immunogenic compositions or of protective vaccines against Bordetella infections as well as antibodies directed against these amino acid sequences, capable of being used for example in immunotherapy. Immunotherapy or serotherapy is especially applicable in children, where appropriate in infants infected with B. pertussis, B. parapertussis or B. bronchiseptica. The invention proposes applications in human medicine or in veterinary medicine.
The subject of the present invention is an amino acid sequence derived from the polypeptide sequence of adenyl cyclase-haemolysin (AC-Hly), characterized in that it is capable of inducing the formation of protective antibodies against an infection by B. pertussis and/or B. parapertussis and/or B. bronchiseptica, and in that it is chosen from the following chains:                a) a sequence comprising the chain of amino acids situated approximately between position 910, preferably 913, and the last C-terminal amino acid of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO:2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO:4), the said sequence comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983;        b) sequence comprising a chain of 6 to 500 amino acids comprising amino acids 385 to 400 of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO: 4);        c) a sequence comprising the chains of amino acids defined above in a) and b), the said chains being either contiguous, or combined via the chain of amino acids naturally present between the chains defined above in a) and b) within the AC-Hly from B. pertussis, B. parapertussis or B. bronchiseptica, or combined via an antigenic sequence derived from a protein different from AC-Hly, the amino acid sequence obtained having a three-dimensional conformation identical or analogous to that of the corresponding polypeptide sequence of AC-Hly from B. pertussis, B. parapertussis or B. bronchiseptica.         
The expression “amino acid sequence derived from the polypeptide sequence of AC-Hly” is understood to mean, within the framework of the invention, a sequence whose amino acids are identical, by their nature or by their linkage, to those of the polypeptide sequence of AC-Hly or are, for some of them, substituted, deleted or added, in a manner such that the immunological properties of AC-Hly are conserved. In particular, such a sequence, derived from the polypeptide sequence of AC-Hly is recognized by antibodies formed in a patient infected with Bordetella, especially with B. pertussis, B. parapertussis or B. bronchiseptica. 
It will be considered, within the framework of the present invention, that the structure or the conformation of AC-Hly is conserved, the amino acid sequence of the invention then having a structure identical or analogous to that of AC-Hly, when the said amino acid sequence is capable, after immunization of a patient or an animal, of inducing a protective immunity against Bordetella infections.
A sequence according to the invention can be obtained by proteolysis of AC-Hly purified from Bordetella. Preferably, this sequence is obtained by chemical synthesis or by genetic engineering techniques.
Thus, to produce an amino acid sequence according to the invention by genetic engineering, plasmids carrying fragments of the CyaA gene will be used.
By way of example, the chemical synthesis may be performed in automatic machines of the Applied Biosystem type. It is also possible to use the technique of Betsou F. et al., 1993, Infect. Immun., 61:3583–3589.
In this manner, it will be possible to prepare a peptide corresponding to the chain of amino acids 385 to 400, or even 385 to 500 of AC-Hly from B. pertussis or to the corresponding chain of AC-Hly from B. parapertussis or from B. bronchiseptica. The amino acid sequence (SEQ ID NO: 2) as well as the nucleotide sequence (SEQ ID NO: 1) of AC-Hly from B. pertussis has been described in Glaser et al, 1988, Molec. Microb. 2-19–30 and is represented in FIG. 5. The amino acid sequence (SEQ ID NO: 4) and the nucleotide sequence (SEQ ID NO: 3) of AC-Hly from B. bronchiseptica is represented in FIG. 6.
The amino acid sequence of AC-Hly from B. parapertussis and the nucleotide sequence encoding AC-Hly can be obtained by conventional techniques from the DNA from a strain of B. parapertussis, for example the strain No. 1 deposited at the CNCM on 2 Dec. 1994 under the No. I-1498.
When amino acid sequence according to the invention is produced by genetic engineering from the DNA of the CyaA genes from B. pertussis, B. parapertussis or B. bronchiseptica, and when it comprises the amino acid in position 983 of AC-Hly from B. pertussis (SEQ ID NO: 2) or a corresponding amino acid of AC-Hly from B. pertussis or B. bronchiseptica (SEQ ID NO: 4), care will be taken to ensure that this sequence is produced in a cellular host also expressing the CyaC gene from the strains identified above. The expression of the CyaC gene allows the modification by addition of a fatty acid necessary to conserve in the amino acid sequence comprising residue 983. The addition of a fatty acid may, by way of example, be a palmitoylation.
Strains which can be used in order to have access to CyaA and CyaC genes are for example B. pertussis HAV deposited at the CNCM on 19 Oct. 1994 under the No. I-1485, B. parapertussis I-1498 and B. bronchiseptica 973S deposited at the CNCM on 12 May 1989 under the No. I-858.
Moreover, the nucleotide sequences encoding AC-Hly from B. pertussis and from B. bronchiseptica are presented in FIGS. 5 and 6 (SEQ ID NOS: 1 and 3) respectively.
The nucleotide sequence of the CyaC gene which activates the CyaA gene has been described in the publication of Barry E. M. et al. (Journal of Bacteriology, January 1991, p. 720–726).
An advantageous sequence within the framework of the present invention is the sequence corresponding to the regions termed modified region and repeat region of AC-Hly (see FIG. 1).
When an antigenic sequence which is heterologous in relation to the polypeptide sequence of AC-Hly is present, it may be a sequence of a bacterial, viral or parasitic pathogenic organism in particular, against which the formation of protective antibodies, for example, is sought.
The expression “protective antibodies against an infection by B. pertussis and/or B. bronchiseptica” is understood to mean antibodies which protect against the lethal and sublethal infections induced by these bacteria, that is to say which protect against the disease and the infection.
Advantageously, the amino acid sequences according to the invention, which are capable of inducing the formation of protective antibodies against the infections designated above, are associated with factors involved in the virulence of Bordetella, and are used for example in the form of polypeptide preparations or of bacterial extracts having the capacity to induce protection against persistence of the bacteria in the host.
An advantageous amino acid sequence within the framework of the invention is a sequence characterized in that it is in the form of a polypeptide having a three-dimensional conformation identical or analogous to that of the corresponding polypeptide sequence of AC-Hly from B. pertussis or from B. parapertussis or from B. bronchiseptica, in that it comprises the chain of amino acids situated between positions 900 approximately, in particular 910, and the last C-terminal amino acid approximately of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO: 4), the said sequence comprising, in addition, a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, in particular at the level of amino acid 983.
This amino acid sequence comprises the modified region and the repeat region of AC-Hly.
Another preferred amino acid sequence according to the invention is a sequence characterized in that it is formed by the chain of amino acids between the amino acid in position 385 approximately and approximately the last C-terminal amino acid of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO: 4), the said sequence comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983.
A subject of the invention is also an amino acid sequence entering within the scope of the definitions given above, formed by the chain of amino acids between the amino acid in position 385 approximately and the amino acid in position 400 approximately of the polypeptide sequence of AC-Hly from B. pertussis (SEQ. ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ. ID NO: 4).
This amino acid sequence advantageously comprises an epitope capable of inducing the formation of protective antibodies against an infection by Bordetella of the B. pertussis, B. parapertussis and B. bronchiseptica types.
According to another embodiment of the invention, the amino acid sequence is characterized in that it is formed by the chain of amino acids between the amino acid in position 385 approximately and the amino acid in position 500 approximately of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO: 4).
Advantageously, this sequence comprising amino acids 385 to 400 or 385 to 500 is presented in a conformation identical or analogous to the conformation which it possesses in the AC-Hly protein from Bordetella. 
The subject of the invention is also amino acid sequences obtained by deletion of polypeptide fragments from the sequence of AC-Hly from Bordetella, whether it is the sequence purified from the bacterium, and in particular from B. pertussis, B. parapertussis or B. bronchiseptica or whether it is a sequence obtained from a recombinant protein r-AC-Hly.
An amino acid sequence thus obtained is advantageously the sequence called ΔCla corresponding to the AC-Hly chain modified by deletion of an ΔCla fragment represented in FIG. 1, corresponding to the chain of amino acids 827 to 887 of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO: 4), the sequence obtained comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983.
Another fragment may be deleted from the AC-Hly sequence in order to form an amino acid sequence according to the invention; this is the polypeptide fragment AH corresponding to the chain of amino acids 385 to 827 of the polypeptide sequence of AC-Hly from B. pertussis (SEQ ID NO: 2) or of a sequence corresponding to the preceding one in B. parapertussis or in B. bronchiseptica (SEQ ID NO: 4), the sequence obtained comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983.
The subject of the invention is also the amino acid sequence forming the “repeat region” of AC-Hly from Bordetella, between the amino acid residues 1000 and 1600 approximately. The repeat region from B. bronchiseptica comprises one repeat less compared to the AC-Hly from B. pertussis. In this regard, the invention relates, for example, to the sequence comprising amino acids 1552 to 1592 of AC-Hly from B. pertussis (SEQ ID NO: 2).
The invention also relates to the nucleotide sequences encoding the amino acid sequences described above.
The genes CyaA, CyaB and partially CyaD from B. bronchiseptica were cloned into the plasmid pFBD2 harboured by E. coli K12XL1 and deposited at the CNCM on 21 Jun. 1995 under the No. I-1601.
The plasmid pFBD2 is obtained by insertion at the BamHI site of the 8 kb fragment from B. bronchiseptica carrying the genes CyaA, CyaB and partially CyaD.
The subject of the invention is moreover polypeptide compositions comprising sequences according to the invention originating from various types of Bordetella. For example, an advantageous polypeptide composition comprises a sequence defined above from B. pertussis or another sequence or several of these sequences from B. parapertussis. 
Another polypeptide composition of the invention comprises, in addition, one or more sequences from B. bronchiseptica. 
According to another embodiment of the invention, a polypeptide composition is characterized in that it comprises one or more sequences defined above from B. parapertussis and one or more sequences from B. bronchiseptica. 
The subject of the invention is also immunogenic compositions characterized in that they comprise one or more sequences defined in the preceding pages.
Advantageously for protection against infection by Bordetella and in particular for protection against persistence of the bacteria in the host, an immunogenic composition comprising the amino acid sequences of the invention may be characterized in that it comprises, in addition, a bacterial extract containing the products of expression of the vrg genes from a strain of Bordetella chosen from B. pertussis, B. parapertussis or B. bronchiseptica or a portion of these expression products, sufficient to induce an immune response in a host to which the extract might be administered.
In addition to the presence of various adhesins and toxins, Bordetellae are characterized by regulation of the expression of the factors involved in their virulence. In other words, Bordetellae undergo phase variations and modulations.
Bordetellae, depending on their environment, can become “avirulent”, that is to say incapable of inducing lethality, an inflammatory reaction and pulmonary lesions in the murine model of respiratory infection. They undergo either a phase modulation or a phase variation. The phase variation is observed at a frequency ranging from 10−3 to 10−6 and is practically reversible. It results in a stoppage of the expression of the toxins and adhesins described above and in the expression of other factors still not well characterized (change of the Phase I “virulent” bacteria to Phase IV “avirulent” bacteria). The phase I and phase IV bacteria have been described by Lacey B. 1960, J. Hyg, 58:57–93. The phase modulation, phenotypically similar to the phase variation, is completely reversible and results in a stoppage of the synthesis of the adhesins and toxins during environmental changes (composition of the culture medium, temperature and the like).
The phase variation and the phase modulation observed in Bordetella are under the control of two regulatory genes, bvg A and bvg S (Arico B et al., 1989, Proc. Natl. Acad. Sci. USA, 86:6671–6675).
The bvg S gene encodes a protein sensitive to the external conditions. This protein modulates, by phosphorylation, the activity of the protein encoded by the bvg A gene, which is, on the one hand, a positive activator of the transcription of the genes encoding the factors for virulence (vag genes for “vir activated genes”) mentioned above (Uhl M. A. and Miller J, 1994, Proc. Natl. Acad. Sci. USA 91:1163–1167), and on the other hand a repressor of the transcription of certain genes (Beattie D. T. et al., J. of Bacteriology, Jan. 93, p. 159–527). The genes whose expression is repressed are called vrg genes for “vir repressed genes” and are yet poorly characterized. It has however been shown that the vrg 6 gene from B. pertussis encodes a protein having a role in the persistence of the bacterium in the host (Beatties D. et al., 1992, Infect. Imm. 60:571–577). In B. bronchiseptica, two proteins encoded by the vrg genes have been characterized: they are proteins of the flagella type (B. bronchiseptica phase I is an immotile bacterium which does not synthesize flagella but which synthesizes adhesins and toxins, and B. bronchiseptica phase IV is a motile bacterium which synthesizes flagella).
The presence, in the immunogenic composition, of a bacterial extract comprising the products of expression of the vrg genes would make it possible to enhance the humoral and/or cellular immune response obtained after infection in vaccinated subjects and would also contribute to the protection against the persistence of the bacterium.
The bacterial extract termed “vrg bacterial extract” which is in question above contains all the constituents of the external membrane of a phase IV bacterium, that is to say of a bacterium not expressing the vag genes, including the LPS endotoxin. This endotoxin may however be eliminated or detoxified.
This extract may be present in the form of a suspension.
The “vrg” bacterial extracts used to carry out the invention are preferably extracts termed “urea extracts”.
A “urea extract” is composed of a mixture of proteins expressed at the surface of the bacterium and which are separated from the bacterium after incubation of the latter with 5 M urea. The “vrg” urea extract contains several proteins as yet not characterized, the flagella and the LPS.
The use of urea extracts allows the production of a vaccine which is cheaper compared with a vaccine which would be obtained from the proteins contained in the extracts, in purified form.
In addition, the inventors have observed that the urea extracts used may induce a T type cell immuno response (lymphoproliferation), thus behaving like the cellular vaccine used up until now.
On the contrary, exclusively acellular compositions are thought not to induce a T response, a reaction which nevertheless occurs in the event of infection.
The vrg urea extracts are respectively prepared from phase IV bacteria. Where appropriate, the phase IV bacteria are replaced with bacteria whose bvg S gene is mutated such that the bacteria express only the proteins encoded by the vrg genes.
The preparation of these extracts is described in detail in the experimental part.
Thus, the invention preferably relates to an immunogenic composition comprising both a vag urea extract from B. pertussis and a vrg urea extract from B. pertussis. 
An appropriate B. pertussis strain for the preparation of these extracts is the HAV strain entering within the framework of the invention and deposited at the CNCM (Collection Nationale de Cultures de Microorganismes in Paris), on 19 Oct. 1994 under the No. I-1485. To prepare the vag urea extract, the HAV strain can be used directly since it is a phase I strain.
On the other hand, the vrg urea extract is obtained from a phase IV strain derived from the phase I strain for example by mutation of the bvg S gene from the bacterium or by culture of the said phase I strain in a medium containing only magnesium sulphate, so as to obtain the expression of the B. pertussis vrg genes alone.
In the same manner and where appropriate to improve the immune response in the host to which the immunogenic composition might be administered, the latter comprises, in addition, one or more adhesins or toxins from B. pertussis, B. parapertussis or B. bronchiseptica, chosen from FHA, AGGs or PRN and PTX or a portion of these proteins, sufficient to induce an immune response in a host to which the extract might be administered.
Advantageously, the amino acid sequences derived from the AC-Hly toxin and, where appropriate, the proteins expressed by the vrg genes are obtained from the HAV strain deposited at the CNCM under the No. I-1485.
Likewise, the amino acid sequences from B. parapertussis used within the framework of the invention are obtained from strain No. 1 deposited at the CNCM under the No. I-1498.
Moreover, the amino acid sequences from the bronchiseptica strain which are used within the framework of the invention may be obtained from the strain 9735 deposited at the CNCM under the No. I-858.
The references given above for the various Bordetella strains are indicated either to give access to the sequence of the proteins and where appropriate to reproduce this sequence by chemical synthesis, or to obtain the amino acid sequences of the invention by proteolysis of the proteins from Bordetella, or to give access to the DNA of the strains and thus to allow the production of the amino acid sequences of the invention by genetic engineering techniques.
The subject of the invention is also a vaccine composition comprising, as active ingredient, an immunogenic composition defined above, in combination with a pharmaceutically acceptable vehicle and, where appropriate, with an adjuvant.
The invention also relates to a process for the preparation of monoclonal antibodies recognizing the AC-Hly from B. pertussis, the AC-Hly from B. parapertussis and the AC-Hly from B. bronchiseptica, comprising the steps of:                immunizing an animal, for example a Balb/c mouse with a peptide comprising the sequence of amino acids 385 to 400 of AC-Hly from B. pertussis (SEQ ID NO: 2), the immunization being, where appropriate, carried out by means of repeated administrations of the peptide;        fusing the spleen cells of the immunized animal with myeloma cells to form a hybridoma;        culturing the hybridoma under conditions allowing the production of antibodies;        recovering the antibodies directed against the sequence of amino acids 385 to 400 of the AC-Hly from B. pertussis.         
The process described above advantageously allows, by using for the immunization an antigen specific for the AC-Hly from B. pertussis, monoclonal antibodies to be obtained which recognize both the AC-Hly from B. pertussis and those from B. parapertussis and from B. bronchiseptica. In addition, such monoclonal antibodies advantageously have protective capacities in relation to the infection of a human or animal host by Bordetella of the B. pertussis, B. parapertussis and/or B. bronchiseptica type. Thus, the monoclonal antibodies obtained by using the process described above can be used for the treatment of patients or animals infected with one or more strains of Bordetella chosen from B. pertussis, B. parapertussis or B. bronchiseptica. 
In general, the subject of the invention is monoclonal antibodies characterized in that they recognize the sequence of amino acids 385 to 400 of the AC-Hly from B. pertussis. 
The invention relates, in this regard, to monoclonal antibodies which recognize an epitope comprising the sequence of amino acids 385–400 of the AC-Hly from B. pertussis, produced by the hybridoma B5–4 deposited at the CNCM on 19 Jun. 1996 under the No. I-1734.
Other advantageous monoclonal antibodies are produced against the sequence of the last 217 amino acids of the AC-Hly from B. pertussis (SEQ ID NO: 2) and are produced by the hybridoma E17-21 deposited at the CNCM on 19 Jun. 1996 under the No. I-1733.
The subject of the invention is also monoclonal antibodies directed against any of the amino acid sequences described above. Monoclonal antibodies directed specifically against the C-terminal sequence of the AC-Hly are among the advantageous antibodies of the invention. Special antibodies are for example directed against a sequence derived from the corresponding chain comprising the last 217 amino acids of the AC-Hly from B. pertussis (SEQ ID NO: 2), especially the amino acids 1488 to 1705 of the AC-Hly from B. pertussis (SEQ ID NO: 2) or the amino acids 1489 to 1706 of the AC-Hly from B. bronchiseptica (SEQ ID NO: 4). These monoclonal antibodies may be prepared by a process analogous to that which is described above for the monoclonal antibodies obtained against the epitope contained in the sequence of amino acids 385 to 400 approximately of the AC-Hly from B. pertussis (SEQ ID NO: 2).
The antibodies of the invention may be humanized, for example, by replacing the hypervariable part of a human immunoglobulin, having no antibody function, with a hypervariable region of a monoclonal immunoglobulin obtained by means of the technique described above.
Techniques which make it possible to humanize antibodies have for example been described by Waldmann T., June 1991, Science, vol. 252, p. 1657–1662; Winter G. et al., 1993, Immunology Today, vol. 14, No. 6, p. 243–246; Carter et al., May 1992, Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285–4289; Singer et al., 1 Apr. 1993, Journal of Immunology, vol. 150, No. 7, p. 2844–2857.
The subject of the invention is also polyclonal sera as obtained by immunization of an animal with an amino acid sequence corresponding to the definitions given above and recovering the antibodies formed which are capable of recognizing the sequences used for the immunization.
Where appropriate, the immunization comprises the administration, with the amino acid sequences, of an adjuvant.
According to a specific embodiment of the invention, the immunization is carried out with one or more amino acid sequences and with an immunogenic composition comprising various antigens of AC-Hly.
The invention also relates to a pharmaceutical composition comprising, as active ingredient, monoclonal antibodies according to the invention.
These antibodies, in particular the antibodies directed against the sequence comprising the amino acids 385 to 400 and/or the antibodies directed against the C-terminal part of the AC-Hly protein, can be used as medicinal product in immuotherapy in a host infected with B. pertussis and/or B. parapertussis and/or B. bronchiseptica. 
Use will be made for example of the monoclonal antibodies produced by the hydridoma B5-4, deposited on Jun. 19, 1996, under the provision of the Budapest Treaty at the National Collection of Cultures of Microorganisms (C.N.C.M.) in Paris, France, and assigned Accession No. I-1734 or by the hybridoma E17-21 deposited at the CNCM under the No. I-1733.
The antibodies according to the invention may also be used as means for analyzing the Bordetella strains collected. The monoclonal antibodies directed against the amino acid sequence 358 to 400 or against the C-terminal sequence of the AC-Hly protein are thus appropriate for the analysis of the B. pertussis strains.
Also entering within the framework of the invention is a process for the in vitro detection of an infection by a Bordetella especially by B. pertussis, B. parapertussis or B. bronchiseptica, characterized in that a sample of biological liquid from a patient or from an animal capable of being infected by Bordetella is brought into contact with monoclonal antibodies defined above or with a polyclonal serum defined above and in that an immunological reaction is detected between the said monoclonal or polyclonal antibodies and bacteria of the genus Bordetella when they are present in the sample.
The detection of infection with a bacterium of the Bordetella genus, especially by B. pertussis, B. parapertussis and B. bronchiseptica, may also be carried out on a sample of a biological fluid from a patient or from an animal by bringing the sample tested into contact with an amino acid sequence corresponding to the definitions above, followed by the detection of an immunological reaction between the said amino acid sequences and the antibodies present in the sample tested.
The invention also relates to a pharmaceutical composition comprising, as active ingredient, nucleotide sequences described above.
The use of the nucleotide sequences in pharmaceutical compositions may be implemented with reference to the technique described by Donnelly et al. (Nature Mèdecine, 1995, vol. 1. No. 6, p. 583–587).